Aminoacyl-tRNA Synthetases (AARS) are enzymes that charge (acylate) tRNAs with amino acids. These charged aminoacyl tRNAs then participate in mRNA translation and protein synthesis. The AARS show high specificity for charging a specific tRNA with the appropriate amino acid, for example valyl-tRNA with valine by valyl-tRNA synthetase or tryptophanyl-tRNA with tryptophan by tryptophanyl-tRNA synthetase. In general, per organism there are at least one AARS for each of the twenty amino acids. There are exceptions however. AARS are ancient enzymes, having functioned in translation since early life evolution. Some have speculated that the earliest aminoacyl-tRNA synthetases were mRNAs, not proteins, with the proteinaceous AARS described here emerging later (Neidhardt et al., (1975) Annu. Rev. Microbiol. 29:215-250). AARS are structurally diverse, although AARSs for some amino acids are more closely related than for others. AARSs are generally divided into two classes, class I and class II based on structural similarity and amino acid preferences (Eriani et al., (1990) Nature 347:203-206).
Plants like all other cellular organisms have aminoacyl-tRNA synthetases. However, a full description of the plant ‘complement’ of aminoacyl-tRNA synthetases has not yet been described. Full-length cDNA, genomic clones, and EST sequences for a variety of plant aminoacyl-tRNA synthetases are known. However, several anticipated aminoacyl-tRNA synthetases have not been discovered.
Because of the central role of protein synthesis in life, any agent that inhibits or disrupts this activity is likely to be toxic. Aminoacyl-tRNA synthetases play a critical role in protein translation by linking genetic nucleic acid information to protein synthesis. Aminoacyl-tRNA synthetases perform this role by “reading” the identity of the different tRNAs and acylating them with the correct cognate amino acid. A large volume of research over several decades has been focused on identifying inhibitors of this process. Inhibitors of aminoacyl-tRNA synthetases have been found to be cytotoxic due to their inhibition of protein synthesis. As such they therefore could be used as herbicides or in aminoacyl-tRNA synthetase selectable marker systems (Lloyd et al., (1995) Nucleic Acid Research 23(15):2882-2892). The genes disclosed herein can serve as the basis for testing whether the encoded aminoacyl-tRNA synthetases are sensitive to known inhibitors or other chemicals.
Biochemical processes are often compartmentalized in regions of cells, such as mitochondria, plastids, and lysosomes. These organelles are key sites for many biochemical pathways. Bioengineering of these processes may require targeting protein products to specific organells. One method to accomplish this involves the addition of an N-terminal prosequence (transit peptide) that directs protein entry into a specific organelle(s). Upon or shortly after transport into the organelle the transit peptide is usually proteolytically removed, and the mature protein is then able to function.
A few plant transit peptides have been shown empirically to be capable of directing fused proteins into specific organelles. However this ability appears to depend upon the structure of the protein being imported and to date it is impossible to predict whether a protein will be imported into an organelle with a given transit peptide. As such, it is advantageous to have a diversity of potential transit peptides from which the most efficient candidate can be chosen to target a protein of interest to an organelle. A number of plant transit peptides are known which direct mature proteins to mitochondria or chloroplast organells. These transit peptides are diverse in structure (length and amino acid sequence) and there is no strong consensus sequence identifying them. In addition, there is no obvious clear relationship between chloroplast targeting and mitochondrial targeting transit sequences. This invention describes a number of chloroplast-targeting and mitochondria-targeting transit peptides for (maize) aminoacyl-tRNA synthetases. These sequences will find utility in directing both aminoacyl-tRNA synthetase and other proteins into these organelles.
Accordingly, the availability of nucleic acid sequences encoding all or a portion of these enzymes would facilitate studies to better understand protein synthesis in plants, provide genetic tools for the manipulation of gene expression, protein targeting to specific organells and provide possible targets for herbicides.